The understanding of the interaction of extraneous molecules with the machinery of a living cell is a central topic for all science and industrial activities where one desires to effect a change on a biological system by treatment with a chemical. This includes basic cellular research, pharmaceutical discovery, toxicology, agricultural sciences and environmental testing. The interaction can be studied at several levels of intricacy, however one is usually interested in relating the gross effect on a biological system with the molecular interactions that cause the effect. The usual mode of approaching this problem has been to identify a chemical that has an affect on an organism, then find the cellular molecule with which the chemical interacts. Alternatively, one can isolate cellular molecules, find chemicals that interact with them either by measuring binding of the two or by measuring a change in the function of the cellular molecule by the chemical. The chemical is then tested for its affect on the cell or organism as a whole. In an industry where discovery of the biological effect of chemicals at the molecular level is central to the discovery of useful compounds, the process of identifying biologically active compounds and their cellular “targets”, becomes a key economic factor. Systems, materials and techniques that increase the efficiency of this process are useful and valuable.
The development of new techniques of measuring interaction between chemicals and biological molecules such as proteins, nucleic acids and membrane lipids has lead to the application of systems with the capacity for assaying many reactions simultaneously. These systems, referred to as high through put (HTP) systems, although automated and rapid, still demand either choosing the type of assay based on the known characteristics of the cellular target, or isolating the cellular target so that a physical interaction between the target and the chemical compound can be measured. Here we disclose a genetic system for generating ordered arrays of hundreds of functional cellular targets which can be used to assay the biological activities of large numbers of chemical compounds, with no prior understanding of the function of the chemical compound, nor prior choice of type of target to be assayed. The system can be used to both determine if a compound has an activity against one of the targets and identify the target.
The genetic system employs the yeast, Saccharomyces cerevisiae. The genetics of the organism are better understood than any other eukaryote and it is relatively easily manipulated genetically. The organism can grow either as a diploid with 16 pairs of homologous chromosomes, or after undergoing sporulation, as a haploid having only one member of each chromosome pair.
Saccharomyces cerevisiae has long served as a useful model for the analysis of eukaryotic gene expression and as a system to study the function of genes isolated from other organisms (heterologous genes) since yeast can be efficiently transformed by DNA molecules consisting of circular or linear plasmids carrying foreign genes under the control of a yeast transcription promoter. In some cases the protein product of the heterologous gene can functionally replace a missing or mutated yeast protein. This type of functional complementation has been used to identify and study genes from more complex organisms, such as higher plants and humans, since yeast can be much more easily grown than higher organisms or their cells. However, it requires that the yeast be made dependent on the foreign gene to grow. Current methods to do this are tedious and require cloning of the yeast gene to be replaced, as well as a complex selection procedure.
The elucidation of the function of many yeast genes followed the completion of the nucleotide sequence in 1996. A systematic study in which each open reading frame (orf) is deleted and the growth the haploid yeast carrying the deletion is measured has thus far revealed that of the some 6200 genes about 20% appear to be essential for growth.
The present invention provides for construction of yeast strains in which selection for haploid cells containing inactivated essential genes allows the simultaneous selection of strains in which the inactivated essential gene is replaced by a heterologous gene which may support growth or other critical cell functions. This invention also provides for the construction of arrays of such strains in which each member of the array contains a heterologous gene upon which that strain is dependent. These arrays in turn serve as platforms for the identification of chemical compounds that affect the function of heterologous genes expressed by individual members of the array. The array is therefore a system that can be used for both determining if a chemical compound is biologically active and identifying the target of the activity.